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D J Phillips

Publications and source records attributed to D J Phillips.

At least 55 records · Page 3Linked to original sources
PubMed1997

Endothelial cell protein S synthesis is upregulated by the complex of IL-6 and soluble IL-6 receptor.

We have recently demonstrated that the proinflammatory cytokine, interleukin-6 (IL-6), could upregulate the production of protein S in the human hepatoma cell line, HepG-2, but not in endothelial cells. In this study, we have demonstrated that the combination of exogenous IL-6 and soluble IL-6 receptor (sIL-6R) could significantly upregulate protein S production in both primary human umbilical vein endothelial cells (HUVEC) and in the immortalized human microvascular endothelial cell line, HMEC-1. The IL-6/sIL-6R complex was also able to rapidly induce tyrosine phosphorylation of the IL-6 transducer, gp130. Neutralizing antibodies directed against either IL-6 or gp130 blocked protein S upregulation by the IL-6/sIL-6R complex. It was also observed that exogenous sIL-6R could also upregulate protein S by forming a complex with IL-6 constitutively produced by the endothelial cell. Two other cytokines which also utilize the gp130 receptor, oncostatin M (OSM) and leukemia inhibitory factor (LIF), were also able to upregulate endothelial cell protein S. This study demonstrates a mechanism that allows endothelial cells to respond to IL-6 and also illustrates the potential importance of circulating soluble receptors in the regulation of the anticoagulation pathway.

Antigens, CD↗
PubMed1997

Guide to evaluation of permanent impairment of the temporomandibular joint. American Academy of Head, Neck and Facial Pain; American Academy of Orofacial Pain; American Academy of Pain Management; American College of Prosthodontists; American Equilibration Society and Society of Occlusal Studies; American Society of Maxillofacial Surgeons; American Society of Temporomandibular Joint Surgeons; International College of Cranio-mandibular Orthopedics; Society for Occlusal Studies.

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Ankylosis↗
PubMed1996

Oligonucleotide ligation assay for detection of the factor V mutation (Arg506-->Gln) causing protein C resistance.

A point mutation in the Factor V (FV) gene at the activated protein C cleavage site, FV Arg (R)506-->FV Gln (Q)506, is the reported molecular basis for resistance to activated protein C (APC-R). This mutation has been reported in approximately 20-50% of individuals with previously unexplained thrombophilia and 3-5% of the general population. We have adapted an oligonucleotide ligation assay (OLA) for nonisotopic detection of the FV:Q506 mutation which permits rapid screening for this mutation. First, the polymerase chain reaction (PCR) was used for target DNA amplification, thus permitting nonisotopic reporters in the DNA analysis. Then thermostable ligase was used for ligation or covalent coupling of adjacent wild-type and mutant oligonucleotide probes which occurs only when the probes are annealed to a matched amplicon. A colorimetric ELISA-based detection assay was then used to capture 5' biotinylated probes in 96-well streptavidin-coated plates and by virtue of ligation, detection of a 3' digoxigenin reporter probe. Following the addition of anti-digoxigenin conjugate and enhanced alkaline phosphatase signal amplification, colorimetric substrate change was measured in an ELISA plate reader. This assay correctly identified FV genotypes of 290 samples.

Base Sequence↗
PubMed1996

TNF-alpha suppresses IL-1 alpha and IL-6 upregulation of C4b-binding protein in HepG-2 hepatoma cells.

C4b-binding protein (C4BP) is a regulatory protein involved in the regulation of the classical pathway of complement and the natural anticoagulant pathway. C4BP is synthesized by the liver, a target organ of the IL-6 proinflammatory cytokine. C4BP is an acute-phase protein and its basal levels may increase by as much as 4-fold during an inflammatory response. IL-6 which plays a major role in the modulation of the acute-phase proteins, including C4BP, also has been shown by our group to significantly increase hepatocyte production of the anticoagulant protein, protein S. In this study, we have examined the role of cytokine combinations on the production of the C4BP regulatory protein in the HepG-2 hepatoma cell line and report that IL-1 alpha and IL-6 in combination as well as IL-1 alpha and Oncostatin M (OSM) were approximately additive in the upregulation of C4BP while IL-6 and OSM were not and that TNF-alpha blocked both IL-1 alpha and IL-6 but not OSM upregulation of C4BP.

Carcinoma, Hepatocellular↗
PubMed1996

TNF-alpha suppresses IL-6 upregulation of protein S in HepG-2 hepatoma cells.

The pathogenesis of disseminated intravascular coagulation (DIC) has, in part, been attributed to the impairment of the natural anticoagulant protein C/protein S pathway. DIC, which frequently occurs during sepsis, has been linked to cytokines that can induce or modulate procoagulant activity. Three of these cytokines, IL-1 alpha, IL-6, and TNF-alpha have been reported to be increased in the early stages of sepsis. In the present study, we have stimulated HepG-2 hepatoma cell cultures with recombinant human IL-1 alpha, IL-6, TNF-alpha, and oncostatin M (OSM). The results demonstrated that TNF-alpha, and to a lesser degree, IL-1 alpha, could significantly suppress IL-6 upregulation of protein S, whereas the effects of OSM was only suppressed by the combination of IL-1 alpha and TNF-alpha. The combination of IL-1 alpha and TNF-alpha also suppressed protein S production below that of control or basal levels. These results indicate that IL-1 alpha and TNF-alpha may play important regulatory roles in coagulation.

Antineoplastic Agents↗
PubMed1996

More basic isoforms of serum gonadotropins during gonadotropin-releasing hormone agonist therapy in pubertal children.

An acute challenge of exogenous GnRH elicits rapidly increased serum gonadotropin levels with qualitative changes to more basic isoforms of both FSH and LH. Chronic GnRH agonist therapy suppresses endogenous gonadotropins, and the serum levels of FSH and LH are low and fairly constant. A possible qualitative change in the gonadotropins during GnRH agonist therapy was investigated by determination of the median charge of the gonadotropin isoforms before and during therapy in 18 pubertal children. Two different GnRH agonists were studied: buserelin, given intranasally or as a sc implant for 1.5-34 months to five girls, aged 7-10 yr, and for 5-6 months to two boys, aged 11-13 yr; and triptorelin, administered as a depot preparation for 3-6 months to four girls, aged 9-12.5 yr, and for 1-24 months to seven boys, aged 10.5-12 yr. FSH and LH in serum and eluates after electrophoresis in 0.10% agarose suspension were measured with sandwich fluoroimmunoassays. The mean serum FSH and LH levels decreased significantly (P < 0.05) in girls during triptorelin therapy, whereas only the FSH level decreased (P < 0.05) in the boys. There were no significant (P > 0.05) changes in serum gonadotropin levels during buserelin therapy. All of the children had more basic serum isoforms of LH, and all but one had more basic forms of FSH during the GnRH agonist treatments. In a girl who had more basic gonadotropin isoforms after treatment with triptorelin for 2 and 6 months, a GnRH challenge elicited the release of still more basic isoforms. The changes in mean median charge to more basic gonadotropin isoforms were highly significant for both busereline (P < 0.01) and triptorelin (P < 0.001) treatment. An increased (P < 0.001) degree of charge heterogeneity was observed for FSH after triptorelin therapy. These findings show that there is a qualitative change in the isoforms of both FSH and LH in serum during GnRH agonist therapy in pubertal children. The changes in charge to more basic gonadotropin isoforms most likely reflect a direct effect at the pituitary level, leading to the synthesis and/or selective release of less sialylated and sulfated isoforms of the gonadotropins. The observed qualitative changes in the gonadotropin isoforms in these pubertal children may be part of the clinical effects of GnRH agonist therapy, leading to an arrest or regression of puberty.

Adolescent↗
PubMed1996

Effects of the Booroola gene (FecB(B)) on bodymass, testis development and hormone concentrations during fetal life.

In female fetuses the Booroola gene (FecB(B)) is known to affect germ cell development and consequently the pattern of ovarian follicular growth during fetal and post-natal life. However in males, the role of this gene during fetal development is unknown. The aims of the study reported here were to examine the effects of the FecB(B) gene on development of male fetuses with respect to body and organ mass for example, pituitary gland, adrenal and mesonephros), testes development, including numbers of germ cells, and also the plasma concentrations or tissue contents of the reproductive hormones (FSH, LH and testosterone) at days 40, 55, 75, 90 and 135 of gestation. The FecB(B) gene was found to influence litter size, bodymass, crown-rump length and testis mass at most stages of gestation. Some effects were also noted on the mesonephros at days 40 and 55 and on the pituitary and adrenal at days 90 or 135 of gestation. However, the FecB(B) gene was not observed to have an effect on the patterns of germ cell development, on pituitary content or plasma concentrations of immunoreactive or bioactive FSH or immunoreactive LH or testicular content of testosterone. When embryo transfer experiments were performed to eliminate the effects of litter size at days 40, 90 and 135 of gestation nearly all of the differences in bodymass, crown-rump length and organ mass disappeared. The only exception to this was at day 90 when bodymass continued to be lighter and crown-rump lengths smaller in the BB/B + fetuses compared with the +2 fetuses; the significance of this finding remains unknown. It is concluded that for Booroola male fetuses there are no direct effects of the FecB(B) gene on pituitary gonadotrophin function or testicular development after sexual differentiation. Moreover, although there may be temporal differences around day 90 of gestation, there are no long-term, direct effects of the FecB(B) gene on total body, adrenal, testis or pituitary mass. Collectively these findings for the male are similar to those for female fetuses except with regard to germ cell development.

Adrenal Glands↗
PubMed1996

Median charge of gonadotrophin isoforms in the pituitary gland and in the circulation of sheep fetuses from mid- to late gestation.

The heterogeneity of LH and FSH in the sheep fetus was studied by determining the median charge of pituitary and circulating isoforms. Pituitary extracts from male and female fetuses at days 75, 95, 120 and 135 of gestation were subjected to agarose suspension electrophoresis. For all fetuses except the day 75 age group, the median mobility of the gonadotrophin isoforms in matching serum samples from the individual fetuses were also determined. LH and FSH in extracts, peripheral samples and column eluates were measured using sensitive and specific sandwich fluoroimmunoassays for ovine gonadotrophins. The median charge of pituitary LH became more basic (P < 0.001) with gestational age, whereas for pituitary FSH more acidic forms (P < 0.001) were present in the older groups. The female fetuses had more basic pituitary isoforms of LH than the males (P < 0.01) between days 95 and 135, and for FSH at day 75 (P < 0.05). In the matching serum samples, the median charge of the LH (P < 0.001) and FSH (P < 0.05) isoforms were more acidic than those in the pituitary gland. No significant effects of age or sex were detected in the median charge of the gonadotrophin isoforms in serum, but in a number of instances the median charge could not be determined due to low serum concentrations which affected the group sizes. These data show that in the sheep fetus LH and FSH are differentially regulated in qualitative as well as quantitative terms, and that the charge of fetal gonadotrophin isoforms changes according to the age and sex of the fetus.

Animals↗
PubMed1996

Follistatin concentrations in male sheep increase following sham castration/castration or injection of interleukin-1 beta.

Plasma follistatin (FS) concentrations were determined after castration (n = 5) or sham castration (n = 4) of mature rams. Both treatments resulted in a prolonged increase in FS between 7 and 19 h after surgery, which returned to pretreatment concentrations by 24 h. Tumour necrosis factor-alpha (TNF-alpha), a sensitive maker of an acute-phase response, was undetectable in plasma, indicating that the FS response was not induced by trauma due to surgery. In a second experiment, injection of castrated rams (n = 4) with ovine recombinant interleukin-1 beta, an acute-phase mediator, resulted in a sustained rise in FS concentrations within 4 h of injection. Plasma TNF-alpha concentrations increased transiently within 1 h of interleukin-1 beta injection, indicating that an acute-phase response had been initiated. Plasma follicle-stimulating hormone (FSH) concentrations were significantly decreased at 8 and 24 h after interleukin-1 beta injection, strongly suggestive of an inhibitory effect of increased FS concentrations on the secretion of FSH. Injection of castrated rams (n = 2) with a control preparation of recombinant interleukin-2 did not induce an acute-phase response, and plasma FS and FSH concentrations were unaffected. These data show that the testis is not a major source of circulating FS, that the increase in circulating FS following sham castration/castration is not due to an acute-phase response, but that conversely FS concentrations are modulated by the acute-phase mediator, interleukin-1 beta.

Acute-Phase Reaction↗
PubMed1996

Orthopedic manifestations of acute pediatric leukemia.

The variety and distribution of skeletal lesions in children with acute lymphoblastic leukemia is rarely seen in other diseases. Skeletal radiographic changes that can occur in a child with acute leukemia include diffuse osteopenia, metaphyseal bands, periosteal new bone formation, geographic osteolysis, osteosclerosis, mixed osteolysis and sclerosis, and permeative destruction. It is important for orthopedic surgeons to recognize the skeletal manifestations of acute leukemia of childhood because the physician who initially evaluates the child will often be an orthopedic surgeon, and a delay in diagnosis has an adverse affect on survival.

Acute Disease↗
PubMed1995

Hemostatic properties of the SV-40 transfected human microvascular endothelial cell line (HMEC-1). A representative in vitro model for microvascular endothelium.

HMEC-1 is a SV-40T transfected human microvascular endothelial cell line that constitutively expresses RNA transcripts for plasminogen activator inhibitor 1 (PAI-1), tissue-type plasminogen activator (t-PA), protein S (PS), von Willebrand factor (vWF), and thrombomodulin. Tissue factor (TF) can be induced in response to stimulation with tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1alpha) and phorbol 12-myristate 13-acetate (PMA). Proteins corresponding to PAI-1, t-PA, protein S and vWF genes were constitutively released in the culture supernatant. This cell line is a model that will be useful to investigate coagulation/fibrinolytic properties of microvascular endothelium.

Basal Metabolism↗
PubMed1995

Effect of chronic daily oral administration of 17 beta-oestradiol and norethisterone on the isoforms of serum gonadotrophins in post-menopausal women.

OBJECTIVE: Chronic treatment with 17 beta-oestradiol (E2) implants has been found to counteract the formation of more acidic isoforms of the gonadotrophins in post-menopausal women. Oral medication with an oestrogen in combination with a progestagen is a common hormone replacement therapy (HRT) in post-menopausal women. The present study investigated the effect of such a therapy on the concentration and charge of the gonadotrophin isoforms in serum. DESIGN: Serum samples were obtained from 20 post-menopausal women, mean age 60 years (range 50-72 years), treated with continuous daily oral medication of 2 mg E2 combined with 1 mg norethisterone acetate (NETA). FSH, LH and E2 in the serum was measured with fluoroimmunoassays. The median charge and charge heterogeneity of the FSH and LH isoforms were determined for each serum by electrophoresis in 0.1% agarose suspension. Sera from 20 post-menopausal women without a history of HRT served as controls. The results were compared with those from previous studies on post-menopausal women treated with E2 implants and on women with normal menstrual cycles. RESULTS: The E2 level in the oral-E2 + NETA treated women was 198-610 pmol/l, within the range expected during the mid-luteal phase of the normal menstrual cycle and similar to that of the group of women with an E2 implant. The mean LH level was similar to that of the luteal phase of the cycle and significantly lower than that of the controls (P < 0.001), the E2 implant group (P < 0.001) and at the follicular phase of the cycle (P < 0.01). The mean FSH level was similar to that of the follicular phase and the E2 implant group but lower than that of the controls (P < 0.001) and higher than at the luteal phase of the cycle (P < 0.01). The mean values for median charge of both FSH and LH were less acidic than those of the controls (P < 0.001) but more acidic than those for the E2 implant group (P < 0.01; P < 0.001) and for different phases of the menstrual cycle (P < 0.05; P < 0.001). The mean degree of charge heterogeneity of FSH was larger (P < 0.01), while that of LH was smaller (P < 0.01), than for the controls. The mean concentrations of SHBG in the oral E2 + NETA group, the E2 implant group and the controls were similar. CONCLUSION: Chronic oral therapy with 2 mg 17 beta-oestradiol combined with 1 mg norethisterone in post-menopausal women efficiently decreased the serum gonadotrophin levels but only partly counteracted the formation of the more acidic isoforms of FSH and LH after menopause. The differences in the charge for both FSH and LH between the E2 implant and the oral E2 + NETA treated groups may be due to the differences in route of administration of E2 or to the effect of norethisterone or both.

Aged↗
PubMed1995

Effects of ovariectomy and FecBB genotype on the median charge and circulating half-life of pituitary FSH isoforms of ewes.

This study investigated the effects of short-term (20 days) ovariectomy, the effects of FSH assay (radioimmunoassay, receptor assay or in vitro bioassay) and of FecBB genotype on the characteristics of pituitary FSH from Booroola ewes. Pituitary extracts were obtained from ovariectomized homozygous carriers (BB) and non-carriers (++, n = 8 per genotype) and ovary-intact controls (n = 4 per genotype). The extracts (n = 4 per genotype per treatment) were subjected to agarose suspension electrophoresis and the eluates were assayed by the three FSH methods. There were significant effects of ovariectomy (P < 0.01) and assay system (P < 0.05) but not of genotype on the median charge of FSH isoforms. The mean +/- SEM migration rates for FSH in intact and ovariectomized ewes were 0.469 +/- 0.006 and 0.439 +/- 0.006 albumin mobility units, respectively (P < 0.01), indicating a shift to more basic isoforms after short-term ovariectomy. When the pituitary extracts were subjected to anion-exchange HPLC, there was a significant (P < 0.01) shift to more basic isoforms in the ovariectomized ewes as shown using agarose electrophoresis, and no gene effects were noted. When the pituitary extracts (n = 4-8 per group) were injected into mature female mice, there were no significant effects of ovariectomy or genotype on the circulating half-lives of the pituitary FSH isoforms. These results indicate that after short-term ovariectomy, the increase in plasma FSH concentrations is accompanied by a shift in the median charge of pituitary FSH isoforms without any observable change in their metabolic clearance rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

IL-6 upregulates protein S expression in the HepG-2 hepatoma cells.

Several pro-inflammatory cytokines have been shown to be important in the modulation of the procoagulant response. However, what role these cytokines may have in the regulation of coagulation inhibitors is poorly understood. While the hepatocyte is a primary site of synthesis for the anticoagulant protein C and S, it is also a major target cell for the proinflammatory cytokine, IL-6. We have found that stimulation of HepG-2 hepatoma cells with IL-6 (5 ng/ ml) significantly increased the production of the anticoagulant cofactor, protein S, in both a time and dose dependent fashion. This increase was seen at both the RNA and protein level. A mouse monoclonal neutralizing antibody to human IL-6 suppressed the IL-6 effect in a concentration dependent fashion. IL-6 also increased the release of the C4b-binding protein but had no effect on protein C production. When combined with either dexamethasone or soluble IL-6 receptor, the IL-6 response was significantly enhanced. Oncostatin M, a functionally related cytokine, had a similar effect while other related cytokines, IL-11 and leukemia inhibitory factor, only had a marginal effect. IL-1, TGF-beta, and TNF-alpha had no significant effect on protein S production. These results indicate that IL-6 may play an important regulatory role in the anti-coagulant pathway.

Animals↗
PubMed1994

Tumor necrosis factor-alpha downregulates protein S secretion in human microvascular and umbilical vein endothelial cells but not in the HepG-2 hepatoma cell line.

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF-alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.

Carcinoma, Hepatocellular↗
PubMed1994

Development of an ELISA for quantification of human protein S in cell culture fluids using commercial polyclonal antisera.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure protein S antigen released into cell culture fluids. We used readily available commercial polyclonal antisera to develop the assay. This assay was sensitive with a detection limit of about 0.086 ng/ml. Between-assay precision (coefficient of variation) at levels of 0.2, 1.1, and 13.9 ng/ml was 14%, 15%, and 11% respectively. Specificity and accuracy were demonstrated from the use of: 1) culture fluids from 3-primary endothelial cell cultures and 7-cell lines known to constitutively produce protein S; 2) 2-cell lines not synthesizing protein S; and 3) from selected samples of normal and protein S deficient plasma. The ELISA described here was about 12-fold more sensitive and 40-fold more cost effective when compared to a commercial ELISA kit. Thus the assay provided a sensitive, specific, precise and economical method useful for the measurement of the nanogram amounts of protein S commonly encountered in cell culture fluids.

Cell Line↗
PubMed1994

Bioactive follicle-stimulating hormone concentrations in plasma during the estrous cycle of the ewe.

Bioactive FSH (B-FSH) concentrations in plasma were determined during the ovine estrous cycle by means of an in vitro bioassay. The concentrations of B-FSH were elevated during and after the preovulatory LH surge and were significantly (p < 0.05) lower during the late-luteal to mid-follicular phases compared with the mid-luteal phase. A significant (p < 0.05) increase in B-FSH was found about 36 h before the LH surge, at a time when the immunoreactive FSH (I-FSH) concentrations were low and unchanged. The plasma B/I ratio for FSH was relatively constant during the luteal phase; it then increased significantly (p < 0.05) before the LH surge and decreased again at the time of the LH surge itself. Pulsar analysis showed that there were 4 peaks of B-FSH throughout the estrous cycle with 2 during the luteal phase, 1 after the LH surge, and the other either during the follicular phase or associated with the LH surge. For I-FSH there were approximately 8 peaks throughout the cycle with 4 during the luteal phase, 2 after the LH surge, and 1 each during the follicular phase and the preovulatory LH surge. There was a weak negative correlation between I-FSH and immunoreactive inhibin (I-inhibin) during most of the estrous cycle, but B-FSH and the B/I ratio were only correlated (negatively) with I-inhibin in the 24 h before the preovulatory LH surge. These findings suggest that there are significant changes in the circulating isoforms of FSH during the ovine estrous cycle that may affect the growth of antral follicles developing towards ovulation.

Animals↗